Abstract: Protein degradation lies at the heart of biological processes from signal transduction to cell cycle regulation. Compromised clearance of misfolded proteins from cells is the leading cause of human diseases such as neurodegenerative disorders. A common theme manifested in neurodegeneration is the accumulation of insoluble protein aggregates in the brain. However, the role of protein aggregation in the pathophysiology of neurodegeneration remains controversial. It remains a formidable task to remove protein aggregates from the affected neurons. In this proposal, we are taking a bold and innovative approach to attacking this exceedingly difficult problem: harnessing the ubiquitin/proteasome system to investigate protein aggregation and neurodegeneration. This proposal builds upon our previous development of methods to engineer single chain ubiquitin ligase CHIP. We successfully established a novel strategy that enables us to alter the substrate binding specificity of CHIP without affecting its ligase activity. Using Huntington's disease (HD) as a model, we propose herein to create recombinant ubiquitin ligases targeting the disease protein huntingtin (Htt) for ubiquitination. Our long-term goal is to define the structural features that determine the ligase activity of engineered ubiquitin ligases, elucidate the molecular mechanisms underlying protein aggregation and neurodegeneration, and evaluate the therapeutic potential of Htt-specific ubiquitin ligases. If successful, the engineered ubiquitin ligases will represent an unprecedented level of control over protein function in somatic cells, which would have direct impact on proteomic research by introducing novel "protein knockout" tools. In addition, the results of this project will provide unique insights into the fundamental cellular and molecular mechanisms underlying protein quality control and the pathophysiology of neurodegeneration. Application of these findings may help to delay or reverse the detrimental effects of neurodegeneration. Ultimately, it will serve as a prototype for the treatment of other neurodegenerative disorders, as well as non-neuronal human diseases. Public Health Relevance: As our population continues to age, neurodegenerative diseases will increase in prevalence and thus pose a daunting challenge to public health worldwide. These truly disastrous disorders include Alzheimer's, Huntington's, Parkinson's, amyotrophic lateral sclerosis and the frontal temporal dementias. This proposal aims to develop novel tools to target the culprits of neurodegeneration, elucidate underlying mechanisms, and provide therapeutic potential to improve the health of these patients.